Death of developing septal cholinergic neurons following NGF withdrawal in vitro: protection by protein synthesis inhibition

Abstract

Fetal septal neurons were grown in vitro under glass coverslips. This sandwich culture method significantly increased general neuronal survival, reduced glial proliferation, and permitted the removal of serum from the growth medium after 5 d in vitro. Thereafter, a simple, and completely defined, medium was used, and the effects of NGF, NGF withdrawal, and protein synthesis inhibition were examined on septal cholinergic neurons. NGF added to septal cultures at the time of plating resulted in a threefold increase in the number of cholinergic neurons seen at 14 d in vitro but had no effect on the survival of non-cholinergic cells. Cholinergic neurons identified by staining for AChE, ChAT, and p75NGFR could be maintained in serum-free, NGF-supplemented medium for over 40 d. When NGF was removed and NGF antibodies added to 14-d-old cultures, less than 30% of cholinergic neurons survived a further 4 d, but when NGF was similarly withdrawn from 35-d-old cultures, over 75% of cholinergic neurons survived. Reapplication of NGF after 3 but not after 12 or more hours of NGF withdrawal from 14-d-old cultures prevented the death of most cholinergic neurons. When NGF was withdrawn from 14-d-old cultures in the presence of the protein synthesis inhibitor cycloheximide, over 75% of the cholinergic neurons survived. These findings suggest that septal cholinergic neurons are dependent on NGF for survival only during a critical period of development and that growth factor-regulated developmental cell death may occur in CNS neurons by activation of programmed cell death requiring protein synthesis.

Dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) protect hippocampal neurons against excitatory amino acid-induced neurotoxicity

“…Under these very different conditions, DHEA had a major attenuating effect on the neurotoxic action of NMDA. We used a standard concentration of NMDA in vitro (1 mM) that was slightly higher than that of some previous studies because the experiments were done on inverted cultures (15). Parallel experiments showed that, although DHEAS also protected neurons in vitro against NMDA, it seemed less potent because significant protection only was observed at Ϸ10 times the lowest effective dose of DHEA (10 nM) used in these experiments.…”

“…The wells were then flooded with culture medium, a total volume of 500 l of DMEM supplemented with B27 (1:50) growth medium (GIBCO͞BRL), streptomycin (25 g/ ml)/penicillin G sodium (10,000 g/ml)͞amphotericin B (0.85%) (Sigma), and 5% fetal calf serum (GIBCO͞BRL). The coverslips were inverted within 24 h to maximize neuronal survival (14,15). On day 5 (plating day is day 0), the cultures in the medium were changed, and the concentration of fetal calf serum was reduced to 1%.…”

Nerve Growth Factor Rapidly Induces Prolonged Acetylcholine Release from Cultured Basal Forebrain Neurons: Differentiation between Neuromodulatory and Neurotrophic Influences

“…The percentage of cholinergic neurons in BF cultures maintained under similar conditions is low (ϳ1%) (Hartikka and Hefti, 1988;Svendsen et al, 1994). However, their unique ability to synthesize ACh makes quantification of supernatant ACh a reliable measure of their neurotransmitter release.…”

“…Regions of BFCN innervation (e.g., hippocampus, cortex) are enriched in NGF (Korsching et al, 1985;Large et al, 1986), and NGF is retrogradely transported by BFCNs (DiStefano et al, 1992), with these neurons expressing TrkA and p75NTR receptors (Koh and Loy, 1989;Holtzman et al, 1992). NGF and TrkA are important for BFCN development, maintenance, and function in vivo (Vantini et al, 1989;Li et al, 1995;Chen et al, 1997;Fagan et al, 1997;Molnar et al, 1998;Debeir et al, 1999;Ruberti et al, 2000), and NGF exposure (days to weeks) enhances cholinergic markers in BF cultures (Hartikka and Hefti, 1988;Takei et al, 1988Takei et al, , 1989Svendsen et al, 1994;Nonner et al, 1996;Pongrac and Rylett, 1998;Oosawa et al, 1999;Auld et al, 2001). The neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) also enhance cholinergic markers (Nonomura et al, 1995;Nonner et al, 1996;Auld et al, 2001).…”

Nerve growth factor rapidly suppresses basal, NMDA-evoked, and AMPA-evoked nitric oxide synthase activity in rat hippocampus in vivo

“…CytC was delivered as a control protein at the same concentration and flow rate. Concentrations of 0.1 ng͞l of NGF previously have been shown to stimulate the maturation of septal cholinergic neurons in vitro (36).…”